Microfluidic cartridge and method of making same

ABSTRACT

The present technology provides for a microfluidic substrate configured to carry out PCR on a number of polynucleotide-containing samples in parallel. The substrate can be a single-layer substrate in a microfluidic cartridge. Also provided are a method of making a microfluidic cartridge comprising such a substrate. Still further disclosed are a microfluidic valve suitable for use in isolating a PCR chamber in a microfluidic substrate, and a method of making such a valve.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/263,208, filed Apr. 28, 2014 and scheduled to issue on Nov. 14, 2017as U.S. Pat. No. 9,815,057, which claims the benefit of U.S. patentapplication Ser. No. 11/940,310, filed Nov. 14, 2007 and issued as U.S.Pat. No. 8,709,787 on Apr. 29, 2014, which claims the benefit of U.S.Provisional Patent Application No. 60/859,284, filed Nov. 14, 2006, andU.S. Provisional Patent Application No. 60/959,437, filed Jul. 13, 2007.The disclosures of all the above-referenced prior applications,publications, and patents are considered part of the disclosure of thisapplication, and are incorporated by reference herein in their entirety.

TECHNICAL FIELD

The technology described herein generally relates to microfluidiccartridges. The technology more particularly relates to microfluidiccartridges that are configured to carry out PCR on nucleotides ofinterest, particularly from several biological samples in parallel,within microfluidic channels in the cartridge, and permit detection ofthose nucleotides.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, many diagnostic analyses can only bedone with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations—often just one in any given urban area. This means that mosthospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss or mix-up. Second, the equipment inquestion is typically not available ‘on-demand’ but instead runs inbatches, thereby delaying the processing time for many samples becausethey must wait for a machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using polymerase chain reaction (PCR) to amplify avector of interest. Once amplified, the presence or absence of anucleotide of interest from the sample needs to be determinedunambiguously. Sample preparation is a process that is susceptible toautomation but is also relatively routinely carried out in almost anylocation. By contrast, steps such as PCR and nucleotide detection havecustomarily only been within the compass of specially trainedindividuals having access to specialist equipment.

There is therefore a need for a method and apparatus of carrying out PCRon prepared biological samples and detecting amplified nucleotides,preferably with high throughput. In particular there is a need for aneasy-to-use device that can deliver a diagnostic result on severalsamples in a short time.

The discussion of the background to the technology herein is included toexplain the context of the technology. This is not to be taken as anadmission that any of the material referred to was published, known, orpart of the common general knowledge as at the priority date of any ofthe claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

The present technology includes methods and devices for detectingpolynucleotides in samples, particularly from biological samples. Inparticular, the technology relates to microfluidic devices that carryout PCR on nucleotides of interest within microfluidic channels, andpermit detection of those nucleotides.

In particular, the present technology provides for a microfluidiccartridge, comprising: a first PCR reaction chamber; a second PCRreaction chamber; a first inlet, in fluid communication with the firstPCR reaction chamber; a second inlet, in fluid communication with thesecond PCR reaction chamber; a first set of microfluidic valvesconfigured to control motion of a sample from the first inlet to thefirst PCR reaction chamber; and a second set of microfluidic valvesconfigured to control motion of a sample from the second inlet to thesecond PCR reaction chamber.

The present technology includes a process for carrying out PCR on aplurality of polynucleotide-containing samples, the method comprising:introducing the plurality of samples into a microfluidic cartridge,wherein the cartridge has a plurality of PCR reaction chambersconfigured to permit thermal cycling of the plurality of samplesindependently of one another; moving the plurality of samples into therespective plurality of PCR reaction chambers; and amplifyingpolynucleotides contained with the plurality of samples, by applicationof successive heating and cooling cycles to the PCR reaction chambers.

The present technology further comprises a number of other embodiments,as set forth herein.

A microfluidic substrate, comprising: a first PCR reaction chamber; asecond PCR reaction chamber; a first inlet, in fluid communication withthe first PCR reaction chamber; a second inlet, in fluid communicationwith the second PCR reaction chamber; a first set of microfluidic valvesconfigured to isolate the first reaction chamber from the first inlet;and a second set of microfluidic valves configured to isolate the secondPCR reaction chamber from the second inlet.

A microfluidic substrate, comprising: a plurality of sample lanes,wherein each of the plurality of sample lanes comprises a microfluidicnetwork having, in fluid communication with one another: an inlet; afirst valve and a second valve; a first channel leading from the inlet,via the first valve, to a reaction chamber; and a second channel leadingfrom the reaction chamber, via the second valve, to a vent.

A microfluidic cartridge having a plurality of microfluidic networks,wherein each of the microfluidic networks, including a PCR reactionchamber, an inlet hole, and the valves for isolating the PCR reactionchamber, is defined in a single substrate.

A method of carrying out PCR independently on a plurality ofpolynucleotide-containing samples, the method comprising: introducingthe plurality of samples in to a microfluidic cartridge, wherein thecartridge has a plurality of PCR reaction chambers configured to permitthermal cycling of the plurality of samples independently of oneanother; moving the plurality of samples into the respective pluralityof PCR reaction chambers; isolating the plurality of PCR reactionchambers; and amplifying polynucleotides contained with the plurality ofsamples, by application of successive heating and cooling cycles to thePCR reaction chambers.

A microfluidic valve, comprising: a first chamber, connected to a firstload channel; a second chamber, connected to a second load channel; anda flow channel, wherein the first and second load channels are eachconnected to the flow channel, and wherein the first and second loadchannels each contain a thermally responsive substance that, uponactuation of the valve, flows into the flow channel thereby sealing it,and wherein the flow channel is constricted along a length either sideof the first and second load channels.

A microfluidic valve, comprising: a chamber, connected to a loadchannel; and a flow channel, wherein the load channel is connected tothe flow channel, and wherein the load channel contains a thermallyresponsive substance that, upon actuation of the valve, flows into theflow channel thereby sealing it, and wherein the flow channel isconstricted along a length either side of the load channel.

A method of making a microfluidic valve, the method comprising:directing a dispensing head over an inlet hole in a microfluidicsubstrate; propelling a quantity of thermally responsive substance fromthe dispensing head into the inlet hole; and maintaining a temperatureof the microfluidic substrate so that the thermally responsive substanceflows by capillary action from the inlet hole into a microfluidicchannel in communication with the inlet hole.

The microfluidic cartridge described herein can be configured for usewith an apparatus comprising: a chamber configured to receive themicrofluidic cartridge; at least one heat source thermally coupled tothe cartridge and configured to apply heat and cooling cycles that carryout PCR on one or more microdroplets of polynucleotide-containing samplein the cartridge; a detector configured to detect presence of one ormore polynucleotides in the one or more samples; and a processor coupledto the detector and the heat source, configured to control heating ofone or more regions of the microfluidic cartridge.

The details of one or more embodiments of the technology are set forthin the accompanying drawings and further description herein. Otherfeatures, objects, and advantages of the technology will be apparentfrom the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plan view of an exemplary multi-lane microfluidiccartridge;

FIG. 2A shows an exemplary multi-lane cartridge;

FIG. 2B shows a portion of an exemplary multi-lane cartridge of FIG. 2A;

FIG. 3 shows a plan of microfluidic circuitry and inlets in an exemplarymulti-lane cartridge;

FIGS. 4A-4C show layer construction, and cross section of an exemplarymicrofluidic cartridge;

FIG. 5 shows a heater array for an exemplary highly-multiplexedmicrofluidic cartridge;

FIGS. 6-9 show various aspects of exemplary highly multiplexedmicrofluidic cartridges;

FIGS. 10A-10C show various aspects of a radially configured highlymultiplexed microfluidic cartridge and associated heater array;

FIG. 11 shows an exemplary microfluidic network in a lane of amulti-lane cartridge such as that for FIG. 1 or 3;

FIGS. 12A-12C show exemplary microfluidic valves, and a gate FIG. 12D;

FIG. 13 shows an exemplary bubble vent;

FIG. 14 shows a cross-section of a portion of a microfluidic cartridge,when in contact with a heater substrate;

FIGS. 15A and 15B show a plan view of heater circuitry adjacent to a PCRreaction chamber; FIG. 15C shows an overlay of an array of heaterelements on an exemplary multi-lane microfluidic cartridge, whereinvarious microfluidic networks are visible;

FIG. 16 shows various cut-away sections that can be used to improvecooling rates during PCR thermal cycling;

FIG. 17 shows a plot of temperature against time during a PCR process,as performed on a microfluidic cartridge as described herein;

FIG. 18 shows an exemplary assembly process for a cartridge as furtherdescribed herein;

FIGS. 19A and 19B show exemplary deposition of wax droplets intomicrofluidic valves;

FIG. 20 shows an exemplary apparatus, a microfluidic cartridge, and aread head contains a detector, as further described herein;

FIG. 21 shows a cross-section of a pipetting head and a cartridge inposition in a microfluidic apparatus;

FIG. 22 shows introduction of a PCR-ready sample into a cartridge,situated in an instrument;

FIG. 23 shows an exemplary 48-lane cartridge;

FIG. 24 shows a heater configuration used for actuating the 48-lane PCRcartridge of FIG. 23;

FIGS. 25A and 25B respectively show an exemplary cartridge and laneconfiguration for a cartridge that permits retrieval of amplifiedsample;

FIG. 26A shows components of a kit, including an exemplary cartridge andreagents; FIG. 26B shows manual action of pipetting a PCR solution intoPCR lanes of the cartridge;

FIG. 27 shows an exemplary cartridge partially removed from a sealedpouch;

FIGS. 28A and 28B show exemplary apparatus for carrying out waxdeposition;

FIGS. 29A-29C show an exemplary 24-lane cartridge in plan view,perspective views, and cross-section, respectively;

FIGS. 30A-30D show aspects of a 96-lane cartridge; and

FIG. 31 shows a real-time PCR trace.

DETAILED DESCRIPTION Microfluidic Cartridge

The present technology comprises a microfluidic cartridge that isconfigured to carry out an amplification, such as by PCR, of one or morepolynucleotides from one or more samples. It is to be understood that,unless specifically made clear to the contrary, where the term PCR isused herein, any variant of PCR including but not limited to real-timeand quantitative, and any other form of polynucleotide amplification isintended to be encompassed. The microfluidic cartridge need not beself-contained and can be designed so that it receives thermal energyfrom one or more heating elements present in an external apparatus withwhich the cartridge is in thermal communication. An exemplary suchapparatus is further described herein; additional embodiments of such asystem are found in U.S. patent application Ser. No. 11/985,577,entitled “Microfluidic System for Amplifying and DetectingPolynucleotides in Parallel”, and filed on even date herewith, thespecification of which is incorporated herein by reference.

By cartridge is meant a unit that may be disposable, or reusable inwhole or in part, and that is configured to be used in conjunction withsome other apparatus that has been suitably and complementarilyconfigured to receive and operate on (such as deliver energy to) thecartridge.

By microfluidic, as used herein, is meant that volumes of sample, and/orreagent, and/or amplified polynucleotide are from about 0.1 μl to about999 μl, such as from 1-100 μl, or from 2-25 μl. Similarly, as applied toa cartridge, the term microfluidic means that various components andchannels of the cartridge, as further described herein, are configuredto accept, and/or retain, and/or facilitate passage of microfluidicvolumes of sample, reagent, or amplified polynucleotide. Certainembodiments herein can also function with nanoliter, volumes (in therange of 10-500 nanoliters, such as 100 nanoliters).

One aspect of the present technology relates to a microfluidic cartridgehaving two or more sample lanes arranged so that analyses can be carriedout in two or more of the lanes in parallel, for example simultaneously,and wherein each lane is independently associated with a given sample.

A sample lane is an independently controllable set of elements by whicha sample can be analyzed, according to methods described herein as wellas others known in the art. A sample lane comprises at least a sampleinlet, and a microfluidic network having one or more microfluidiccomponents, as further described herein.

In various embodiments, a sample lane can include a sample inlet port orvalve, and a microfluidic network that comprises, in fluidiccommunication one or more components selected from the group consistingof: at least one thermally actuated valve, a bubble removal vent, atleast one thermally actuated pump, a gate, mixing channel, positioningelement, microreactor, a downstream thermally actuated valve, and a PCRreaction chamber. The sample inlet valve can be configured to accept asample at a pressure differential compared to ambient pressure ofbetween about 70 and 100 kilopascals.

The cartridge can therefore include a plurality of microfluidicnetworks, each network having various components, and each networkconfigured to carry out PCR on a sample in which the presence or absenceof one or more polynucleotides is to be determined.

A multi-lane cartridge is configured to accept a number of samples inseries or in parallel, simultaneously or consecutively, in particularembodiments 12 samples, wherein the samples include at least a firstsample and a second sample, wherein the first sample and the secondsample each contain one or more polynucleotides in a form suitable foramplification. The polynucleotides in question may be the same as, ordifferent from one another, in different samples and hence in differentlanes of the cartridge. The cartridge typically processes each sample byincreasing the concentration of a polynucleotide to be determined and/orby reducing the concentration of inhibitors relative to theconcentration of polynucleotide to be determined.

The multi-lane cartridge comprises at least a first sample lane having afirst microfluidic network and a second lane having a secondmicrofluidic network, wherein each of the first microfluidic network andthe second microfluidic network is as elsewhere described herein, andwherein the first microfluidic network is configured to amplifypolynucleotides in the first sample, and wherein the second microfluidicnetwork is configured to amplify polynucleotides in the second sample.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source to a sample mixture comprisingPCR reagent and neutralized polynucleotide sample under thermal cyclingconditions suitable for creating PCR amplicons from the neutralizedpolynucleotide sample.

At least the external heat source may operate under control of acomputer processor, configured to execute computer readable instructionsfor operating one or more components of each sample lane, independentlyof one another, and for receiving signals from a detector that measuresfluorescence from one or more of the PCR reaction chambers.

For example, FIG. 1 shows a plan view of a microfluidic cartridge 100containing twelve independent sample lanes 101 capable of simultaneousor successive processing. The microfluidic network in each lane istypically configured to carry out amplification, such as by PCR, on aPCR-ready sample, such as one containing nucleic acid extracted from asample using other methods as further described herein. A PCR-readysample is thus typically a mixture comprising the PCR reagents and theneutralized polynucleotide sample, suitable for subjecting to thermalcycling conditions that create PCR amplicons from the neutralizedpolynucleotide sample. For example, a PCR-ready sample can include a PCRreagent mixture comprising a polymerase enzyme, a positive controlplasmid, a fluorogenic hybridization probe selective for at least aportion of the plasmid and a plurality of nucleotides, and at least oneprobe that is selective for a polynucleotide sequence. Exemplary probesare further described herein. Typically, the microfluidic network isconfigured to couple heat from an external heat source with the mixturecomprising the PCR reagent and the neutralized polynucleotide sampleunder thermal cycling conditions suitable for creating PCR ampliconsfrom the neutralized polynucleotide sample.

In various embodiments, the PCR reagent mixture can include a positivecontrol plasmid and a plasmid fluorogenic hybridization probe selectivefor at least a portion of the plasmid, and the microfluidic cartridgecan be configured to allow independent optical detection of thefluorogenic hybridization probe and the plasmid fluorogenichybridization probe.

In various embodiments, the microfluidic cartridge can accommodate anegative control polynucleotide, wherein the microfluidic network can beconfigured to independently carry out PCR on each of a neutralizedpolynucleotide sample and a negative control polynucleotide with the PCRreagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide. Eachlane of a multi-lane cartridge as described herein can perform tworeactions when used in conjunction with two fluorescence detectionsystems per lane. A variety of combinations of reactions can beperformed in the cartridge, such as two sample reactions in one lane, apositive control and a negative control in two other lanes; or a samplereaction and an internal control in one lane and a negative control in aseparate lane.

FIG. 2A shows a perspective view of a portion of an exemplarymicrofluidic cartridge 200 according to the present technology. FIG. 2Bshows a close-up view of a portion of the cartridge 200 of FIG. 2Aillustrating various representative components. The cartridge 200 may bereferred to as a multi-lane PCR cartridge with dedicated sample inlets202. For example sample inlet 202 is configured to accept a liquidtransfer member (not shown) such as a syringe, a pipette, or a PCR tubecontaining a PCR ready sample. More than one inlet 202 is shown in FIGS.2A, 2B, wherein one inlet operates in conjunction with a single samplelane. Various components of microfluidic circuitry in each lane are alsovisible. For example, microvalves 204, and 206, and hydrophobic vents208 for removing air bubbles, are parts of microfluidic circuitry in agiven lane. Also shown is an ultrafast PCR reactor 210, which, asfurther described herein, is a microfluidic channel in a given samplelane that is long enough to permit PCR to amplify polynucleotidespresent in a sample. Above each PCR reactor 210 is a window 212 thatpermits detection of fluorescence from a fluorescent substance in PCRreactor 210 when a detector is situated above window 212. It is to beunderstood that other configurations of windows are possible including,but not limited to, a single window that straddles each PCR reactoracross the width of cartridge 200.

In preferred embodiments, the multi-sample cartridge has a sizesubstantially the same as that of a 96-well plate as is customarily usedin the art. Advantageously, then, such a cartridge may be used withplate handlers used elsewhere in the art.

The sample inlets of adjacent lanes are reasonably spaced apart from oneanother to prevent any contamination of one sample inlet from anothersample when a user introduces a sample into any one cartridge. In anembodiment, the sample inlets are configured so as to prevent subsequentinadvertent introduction of sample into a given lane after a sample hasalready been introduced into that lane. In certain embodiments, themulti-sample cartridge is designed so that a spacing between thecentroids of sample inlets is 9 mm, which is an industry-recognizedstandard. This means that, in certain embodiments the center-to-centerdistance between inlet holes in the cartridge that accept samples fromPCR tubes, as further described herein, is 9 mm. The inlet holes can bemanufactured conical in shape with an appropriate conical angle so thatindustry-standard pipette tips (2 μl, 20 μl, 200 μl, volumes, etc.) fitsnugly therein. The cartridge herein may be adapted to suit other,later-arising, industry standards not otherwise described herein, aswould be understood by one of ordinary skill in the art.

FIG. 3 shows a plan view of an exemplary microfluidic cartridge 300having 12 sample lanes. The inlet ports 302 in this embodiment have a 6mm spacing, so that, when used in conjunction with an automated sampleloader having 4 heads, spaced equidistantly at 18 mm apart, the inletscan be loaded in three batches of four inlets: e.g., inlets 1, 4, 7, and10 together, followed by 2, 5, 8, and 11, then finally 3, 6, 9, and 12,wherein the 12 inlets are numbered consecutively from one side of thecartridge to the other as shown.

A microfluidic cartridge as used herein may be constructed from a numberof layers. Accordingly, one aspect of the present technology relates toa microfluidic cartridge that comprises a first, second, third, fourth,and fifth layers wherein one or more layers define a plurality ofmicrofluidic networks, each network having various components configuredto carry out PCR on a sample in which the presence or absence of one ormore polynucleotides is to be determined. In various embodiments, one ormore such layers are optional.

FIGS. 4A-C show various views of a layer structure of an exemplarymicrofluidic cartridge comprising a number of layers, as furtherdescribed herein. FIG. 4A shows an exploded view; FIG. 4B shows aperspective view; and FIG. 4C shows a cross-sectional view of a samplelane in the exemplary cartridge. Referring to FIGS. 4A-C, an exemplarymicrofluidic cartridge 400 includes first 420, second 422, third 424,fourth 426, and fifth layers in two non-contiguous parts 428, 430 (asshown) that enclose a microfluidic network having various componentsconfigured to process multiple samples in parallel that include one ormore polynucleotides to be determined.

Microfluidic cartridge 400 can be fabricated as desired. The cartridgecan include a microfluidic substrate layer 424, typically injectionmolded out of a plastic, such as a zeonor plastic (cyclic olefinpolymer), having a PCR channel and valve channels on a first side andvent channels and various inlet holes, including wax loading holes andliquid inlet holes, on a second side (disposed toward hydrophobic ventmembrane 426). It is advantageous that all the microfluidic networkdefining structures, such as PCR reactors, valves, inlet holes, and airvents, are defined on the same single substrate 424. This attributefacilitates manufacture and assembly of the cartridge. Additionally, thematerial from which this substrate is formed is rigid or non-deformable,non-venting to air and other gases, and has a low autofluorescence tofacilitate detection of polynucleotides during an amplification reactionperformed in the microfluidic circuitry defined therein. Rigidity isadvantageous because it facilitates effective and uniform contact with aheat unit as further described herein. Use of a non-venting material isalso advantageous because it reduces the likelihood that theconcentration of various species in liquid form will change duringanalysis. Use of a material having low auto-fluorescence is alsoimportant so that background fluorescence does not detract frommeasurement of fluorescence from the analyte of interest.

The cartridge can further include, disposed on top of the substrate 424,an oleophobic/hydrophobic vent membrane layer 426 of a porous material,such as 0.2 to 1.0 micron pore-size membrane of modifiedpolytetrafluorethylene, the membrane being typically between about 25and about 100 microns thick, and configured to cover the vent channelsof microfluidic substrate 424, and attached thereto using, for example,heat bonding.

Typically, the microfluidic cartridge further includes a layer 428, 430of polypropylene or other plastic label with pressure sensitive adhesive(typically between about 50 and 150 microns thick) configured to sealthe wax loading holes of the valves in substrate 424, trap air used forvalve actuation, and serve as a location for operator markings. In FIG.4A, this layer is shown in two separate pieces, 428,430, though it wouldbe understood by one of ordinary skill in the art that a single piecelayer would be appropriate.

In various embodiments, the label is a computer-readable label. Forexample, the label can include a bar code, a radio frequency tag or oneor more computer-readable characters. The label can be formed of amechanically compliant material. For example, the mechanically compliantmaterial of the label can have a thickness of between about 0.05 andabout 2 millimeters and a Shore hardness of between about 25 and about100. The label can be positioned such that it can be read by a sampleidentification verifier as further described herein.

The cartridge can further include a heat sealable laminate layer 422(typically between about 100 and about 125 microns thick) attached tothe bottom surface of the microfluidic substrate 424 using, for example,heat bonding. This layer serves to seal the PCR channels and ventchannels in substrate 424. The cartridge can further include a thermalinterface material layer 420 (typically about 125 microns thick),attached to the bottom of the heat sealable laminate layer using, forexample, pressure sensitive adhesive. The layer 420 can be compressibleand have a higher thermal conductivity than common plastics, therebyserving to transfer heat across the laminate more efficiently.Typically, however, layer 420 is not present.

The application of pressure to contact the cartridge to the heater of aninstrument that receives the cartridge generally assists in achievingbetter thermal contact between the heater and the heat-receivable partsof the cartridge, and also prevents the bottom laminate structure fromexpanding, as would happen if the PCR channel was only partially filledwith liquid and the air entrapped therein would be thermally expandedduring thermocycling.

In use, cartridge 400 is typically thermally associated with an array ofheat sources configured to operate the components (e.g., valves, gates,actuators, and processing region 410) of the device. Exemplary suchheater arrays are further described herein. Additional embodiments ofheater arrays are described in U.S. patent application Ser. No.11/940,315, entitled “Heater Unit for Microfluidic Diagnostic System”and filed on even date herewith, the specification of which isincorporated herein by reference in its entirety. In some embodiments,the heat sources are controlled by a computer processor and actuatedaccording to a desired protocol. Processors configured to operatemicrofluidic devices are described in, e.g., U.S. application Ser. No.09/819,105, filed Mar. 28, 2001, which application is incorporatedherein by reference.

In various embodiments, during transport and storage, the microfluidiccartridge can be further surrounded by a sealed pouch. The microfluidiccartridge can be sealed in the pouch with an inert gas. The microfluidiccartridge can be disposable for example after one or more of its samplelanes have been used.

HIGHLY MULTIPLEXED EMBODIMENTS

Embodiments of the cartridge described herein may be constructed thathave high-density microfluidic circuitry on a single cartridge thatthereby permit processing of multiple samples in parallel, or insequence, on a single cartridge. Preferred numbers of such multiplesamples include 20, 24, 36, 40, 48, 50, 60, 64, 72, 80, 84, 96, and 100,but it would be understood that still other numbers are consistent withthe apparatus and cartridge herein, where deemed convenient andpractical.

Accordingly, different configurations of lanes, sample inlets, andassociated heater networks than those explicitly depicted in the FIGsand examples that can facilitate processing such numbers of samples on asingle cartridge are within the scope of the instant disclosure.Similarly, alternative configurations of detectors and heating elementsfor use in conjunction with such a highly multiplexed cartridge are alsowithin the scope of the description herein.

It is also to be understood that the microfluidic cartridges describedherein are not to be limited to rectangular shapes, but can includecartridges having circular, elliptical, triangular, rhombohedral,square, and other shapes. Such shapes may also be adapted to includesome irregularity, such as a cut-out, to facilitate placement in acomplementary apparatus as further described herein.

In an exemplary embodiment, a highly multiplexed cartridge has 48 samplelanes, and permits independent control of each valve in each lane bysuitably configured heater circuitry, with 2 banks of thermocyclingprotocols per lane, as shown in FIG. 5. In the embodiment in FIG. 5, theheaters (shown superimposed on the lanes) are arranged in three arrays502, 504, with 506, and 508. The heaters are themselves disposed withinone or more substrates. Heater arrays 502, 508 in two separate glassregions only apply heat to valves in the microfluidic networks in eachlane. Because of the low thermal conductivity of glass, the individualvalves may be heated separately from one another. This permits samplesto be loaded into the cartridge at different times, and passed to thePCR reaction chambers independently of one another. The PCR heaters 504,506 are mounted on a silicon substrate—and are not readily heatedindividually, but thereby permit batch processing of PCR samples, wheremultiple samples from different lanes are amplified by the same set ofheating/cooling cycles. It is preferable for the PCR heaters to bearranged in 2 banks (the heater arrays 506 on the left and right 508 arenot in electrical communication with one another), thereby permitting aseparate degree of sample control.

FIG. 6 shows a representative 48-sample cartridge 600 compatible withthe heater arrays of FIG. 5, and having a configuration of inlets 602different to that depicted o other cartridges herein. The inletconfiguration is exemplary and has been designed to maximize efficiencyof space usage on the cartridge. The inlet configuration can becompatible with an automatic pipetting machine that has dispensing headssituated at a 9 mm spacing. For example, such a machine having 4 headscan load 4 inlets at once, in 12 discrete steps, for the cartridge ofFIG. 6. Other configurations of inlets though not explicitly describedor depicted are compatible with the technology described herein.

FIG. 7 shows, in close up, an exemplary spacing of valves 702, channels704, and vents 706, in adjacent lanes 708 of a multi-sample microfluidiccartridge for example as shown in FIG. 6.

FIGS. 8 and 9 show close-ups of, respectively, heater arrays 804compatible with, and inlets 902 on, the exemplary cartridge shown inFIG. 7.

FIGS. 10A and 10B show various views of an embodiment of aradially-configured highly-multiplexed cartridge, having a number ofinlets 1002, microfluidic lanes 1004, valves 1005, and PCR reactionchambers 1006. FIG. 10C shows an array of heater elements 1008compatible with the cartridge layout of FIG. 10A.

The various embodiments shown in FIGS. 5-10C are compatible with liquiddispensers, receiving bays, and detectors that are configureddifferently from the other specific examples described herein.

During the design and manufacture of highly multiplexed cartridges,photolithographic processing steps such as etching, holedrilling/photo-chemical drilling/sand-blasting/ion-milling processesshould be optimized to give well defined holes and microchannel pattern.Proper distances between channels should be identified and maintained toobtain good bonding between the microchannel substrate and the heatconducting substrate layer. In particular, it is desirable that minimaldistances are maintained between pairs of adjacent microchannels topromote, reliable bonding of the laminate in between the channels.

The fabrication by injection molding of these complicated microfluidicstructures having multiple channels and multiple inlet holes entailsproper consideration of dimensional repeatability of these structuresover multiple shots from the injection molding master pattern. Properconsideration is also attached to the placement of ejector pins to pushout the structure from the mold without causing warp, bend or stretchingof it. For example, impression of the ejector pins on the microfluidicsubstrate should not sink into the substrate thereby preventingplanarity of the surface of the cartridge. The accurate placement ofvarious inlet holes (such as sample inlet holes, valve inlet holes andvent holes) relative to adjacent microfluidic channels is also importantbecause the presence of these holes can cause knit-lines to form thatmight cause unintended leak from a hole to a microchannel. Highlymultiplexed microfluidic substrates may be fabricated in other materialssuch as glass, silicon.

The size of the substrate relative to the number of holes is also factorduring fabrication because it is easy to make a substrate having just asimple microfluidic network with a few holes (maybe fewer than 10 holes)and a few microchannels, but making a substrate having over 24, or over48, or over 72 holes, etc., is more difficult.

Microfluidic Networks

Particular components of exemplary microfluidic networks are furtherdescribed herein.

Channels of a microfluidic network in a lane of cartridge typically haveat least one sub-millimeter cross-sectional dimension. For example,channels of such a network may have a width and/or a depth of about 1 mmor less (e.g., about 750 microns or less, about 500 microns, or less,about 250 microns or less).

FIG. 11 shows a plan view of a representative microfluidic circuit foundin one lane of a multi-lane cartridge such as shown in FIGS. 2A and 2B.It would be understood by one skilled in the art that otherconfigurations of microfluidic network would be consistent with thefunction of the cartridges and apparatus described herein. In operationof the cartridge, in sequence, sample is introduced through liquid inlet202, optionally flows into a bubble removal vent channel 208 (whichpermits adventitious air bubbles introduced into the sample duringentry, to escape), and continues along a channel 216. Typically, whenusing a robotic dispenser of liquid sample, the volume is dispensedaccurately enough that formation of bubbles is not a significantproblem, and the presence of vent channel 208 is not necessary. Thus, incertain embodiments, the bubble removal vent channel 208 is not presentand sample flows directly into channel 216. Throughout the operation ofcartridge 200, the fluid is manipulated as a microdroplet (not shown inFIG. 11). Valves 204 and 206 are initially both open, so that amicrodroplet of sample-containing fluid can be pumped into PCR reactorchannel 210 from inlet hole 202 under influence of force from the sampleinjection operation. Upon initiating of processing, the detector presenton top of the PCR reactor 210 checks for the presence of liquid in thePCR channel, and then valves 204 and 206 are closed to isolate the PCRreaction mix from the outside. In one embodiment, the checking of thepresence of liquid in the PCR channel is by measuring the heat ramprate, such as by one or more temperature sensors in the heating unit. Achannel with liquid absent will heat up faster than one in which, e.g.,a sample, is present.

Both valves 204 and 206 are closed prior to thermocycling to prevent orreduce any evaporation of liquid, bubble generation, or movement offluid from the PCR reactor. End vent 214 is configured to prevent a userfrom introducing an excess amount of liquid into the microfluidiccartridge, as well as playing a role of containing any sample fromspilling over to unintended parts of the cartridge. A user may inputsample volumes as small as an amount to fill the region from the bubbleremoval vent (if present) to the middle of the microreactor, or up tovalve 204 or beyond valve 204. The use of microvalves prevents both lossof liquid or vapor thereby enabling even a partially filled reactor tosuccessfully complete a PCR thermocycling reaction.

The reactor 210 is a microfluidic channel that is heated through aseries of cycles to carry out amplification of nucleotides in thesample, as further described herein, and according to amplificationprotocols known to those of ordinary skill in the art. The inside wallsof the channel in the PCR reactor are typically made very smooth andpolished to a shiny finish (for example, using a polish selected fromSPI A1, SPI A2, SPI A3, SPI B1, or SPI B2) during manufacture. This isin order to minimize any microscopic quantities of air trapped in thesurface of the PCR channel, which would causing bubbling during thethermocycling steps. The presence of bubbles especially in the detectionregion of the PCR channel could also cause a false or inaccurate readingwhile monitoring progress of the PCR. Additionally, the PCR channel canbe made shallow such that the temperature gradient across the depth ofthe channel is minimized.

The region of the cartridge 212 above PCR reactor 210 is a thinned downsection to reduce thermal mass and autofluorescence from plastic in thecartridge. It permits a detector to more reliably monitor progress ofthe reaction and also to detect fluorescence from a probe that binds toa quantity of amplified nucleotide. Exemplary probes are furtherdescribed herein. The region 212 can be made of thinner material thanthe rest of the cartridge so as to permit the PCR channel to be moreresponsive to a heating cycle (for example, to rapidly heat and coolbetween temperatures appropriate for denaturing and annealing steps),and so as to reduce glare, autofluorescence, and undue absorption offluorescence.

After PCR has been carried out on a sample, and presence or absence of apolynucleotide of interest has been determined, it is preferred that theamplified sample remains in the cartridge and that the cartridge iseither used again (if one or more lanes remain unused), or disposed of.Should a user wish to run a post amplification analysis, such as gelelectrophoresis, the user may pierce a hole through the laminate of thecartridge, and recover an amount—typically about 1.5 microliter—of PCRproduct. The user may also place the individual PCR lane on a specialnarrow heated plate, maintained at a temperature to melt the wax in thevalve, and then aspirate the reacted sample from the inlet hole of thatPCR lane.

In various embodiments, the microfluidic network can optionally includeat least one reservoir configured to contain waste.

Table 1 outlines typical volumes, pumping pressures, and operation timesassociated with various components of a microfluidic cartridge describedherein.

TABLE 1 Displacement Operation Pumping Pressure Volume Time of OperationMoving valve ~1-2 psi <1 μl 5-15 seconds wax plugs Operation Pump UsedPump Design Pump Actuation Moving valve Thermopneumatic 1 μl of trappedHeat trapped air to wax plugs pump air ~70-90 C.

Valves

A valve (sometimes referred to herein as a microvalve) is a component incommunication with a channel, such that the valve has a normally openstate allowing material to pass along a channel from a position on oneside of the valve (e.g., upstream of the valve) to a position on theother side of the valve (e.g., downstream of the valve). Upon actuationof the valve, the valve transitions to a closed state that preventsmaterial from passing along the channel from one side of the valve tothe other. For example, in one embodiment, a valve can include a mass ofa thermally responsive substance (TRS) that is relatively immobile at afirst temperature and more mobile at a second temperature. The first andsecond temperatures are insufficiently high to damage materials, such aspolymer layers of a microfluidic cartridge in which the valve issituated. A mass of TRS can be an essentially solid mass or anagglomeration of smaller particles that cooperate to obstruct thepassage when the valve is closed. Examples of TRS's include a eutecticalloy (e.g., a solder), wax (e.g., an olefin), polymers, plastics, andcombinations thereof. The TRS can also be a blend of variety ofmaterials, such as an emulsion of thermoelastic polymer blended with airmicrobubbles (to enable higher thermal expansion, as well as reversibleexpansion and contraction), polymer blended with expancel material(offering higher thermal expansion), polymer blended with heatconducting microspheres (offering faster heat conduction and hence,faster melting profiles), or a polymer blended with magneticmicrospheres (to permit magnetic actuation of the meltedthermoresponsive material).

Generally, for such a valve, the second temperature is less than about90° C. and the first temperature is less than the second temperature(e.g., about 70° C. or less). Typically, a chamber is in gaseouscommunication with the mass of TRS. The valve is in communication with asource of heat that can be selectively applied to the chamber of air andto the TRS. Upon heating gas (e.g., air) in the chamber and heating themass of TRS to the second temperature, gas pressure within the chamberdue to expansion of the volume of gas, forces the mass to move into thechannel, thereby obstructing material from passing therealong.

An exemplary valve is shown in FIG. 12A. The valve of FIG. 12A has twochambers of air 1203, 1205 in contact with, respectively, each of twochannels 1207, 1208 containing TRS. The air chambers also serve asloading ports for TRS during manufacture of the valve, as furtherdescribed herein. In order to make the valve sealing very robust andreliable, the flow channel 1201 (along which, e.g., sample passes) atthe valve junction is made narrow (typically 150 μm wide, and 150 μmdeep or narrower), and the constricted portion of the flow channel ismade at least 0.5 or 1 mm long such that the TRS seals up a long narrowchannel thereby reducing any leakage through the walls of the channel.In the case of a bad seal, there may be leakage of fluid around walls ofchannel, past the TRS, when the valve is in the closed state. In orderto minimize this, the flow channel is narrowed and elongated as much aspossible. In order to accommodate such a length of channel on acartridge where space may be at a premium, the flow channel canincorporate one or more curves 1209 as shown in FIG. 12A. The valveoperates by heating air in the TRS-loading port, which forces the TRSforwards into the flow-channel in a manner so that it does not come backto its original position. In this way, both air and TRS are heatedduring operation.

In various other embodiments, a valve for use with a microfluidicnetwork in a microfluidic cartridge herein can be a bent valve as shownin FIG. 12B. Such a configuration reduces the footprint of the valve andhence reduces cost per part for highly dense microfluidic cartridges. Asingle valve loading hole 1211 is positioned in the center, that servesas an inlet for thermally responsive substance. The leftmost vent 1213can be configured to be an inlet for, e.g., sample, and the rightmostvent 1215 acts as an exit for, e.g., air. This configuration can be usedas a prototype for testing such attributes as valve and channel geometryand materials.

In various other embodiments, a valve for use with a microfluidicnetwork can include a curved valve as shown in FIG. 12C, in order toreduce the effective cross-section of the valve, thereby enablingmanufacture of cheaper dense microfluidic devices. Such a valve canfunction with a single valve loading hole and air chamber 1221 insteadof a pair as shown in FIG. 12A.

Gates

FIG. 12D shows an exemplary gate as may optionally be used in amicrofluidic network herein. A gate can be a component that can have aclosed state that does not allow material to pass along a channel from aposition on one side of the gate to another side of the gate, and anopen state that does allow material to pass along a channel from aposition on one side of the gate to another side of the gate. Actuationof an open gate can transition the gate to a closed state in whichmaterial is not permitted to pass from one side of the gate (e.g.,upstream of the gate) to the other side of the gate (e.g., downstream ofthe gate). Upon actuation, a closed gate can transition to an open statein which material is permitted to pass from one side of the gate (e.g.,upstream of the gate) to the other side of the gate (e.g., downstream ofthe gate).

In various embodiments, a microfluidic network can include a narrow gate380 as shown in FIG. 12D where a gate loading channel 382 used forloading wax from a wax loading hole 384 to a gate junction 386 can benarrower (e.g., approximately 150 μm wide and 100 microns deep). Anupstream channel 388 as well as a downstream channel 390 of the gatejunction 386 can be made wide (e.g., ˜500 μm) and deep (e.g., ˜500 μm)to help ensure the wax stops at the gate junction 386. The amount ofgate material melted and moved out of the gate junction 386 may beminimized for optimal gate 380 opening. As an off-cartridge heater maybe used to melt the thermally responsive substance in gate 380, amisalignment of the heater could cause the wax in the gate loadingchannel 382 to be melted as well. Therefore, narrowing the dimension ofthe loading channel may increase reliability of gate opening. In thecase of excessive amounts of wax melted at the gate junction 386 andgate loading channel 382, the increased cross-sectional area of thedownstream channel 390 adjacent to the gate junction 386 can prevent waxfrom clogging the downstream channel 390 during gate 380 opening. Thedimensions of the upstream channel 388 at the gate junction 386 can bemade similar to the downstream channel 390 to ensure correct wax loadingduring gate fabrication.

In various embodiments, the gate can be configured to minimize theeffective area or footprint of the gate within the network and thus bentgate configurations, although not shown herein are consistent with theforegoing description.

Vents

In various embodiments, the microfluidic network can include at leastone hydrophobic vent in addition to an end vent. A vent is a generaloutlet (hole) that may or may not be covered with a hydrophobicmembrane. An exit hole is an example of a vent that need not be coveredby a membrane.

A hydrophobic vent (e.g., a vent in FIG. 13) is a structure that permitsgas to exit a channel while limiting (e.g., preventing) quantities ofliquid from exiting the channel. Typically, hydrophobic vents include alayer of porous hydrophobic material (e.g., a porous filter such as aporous hydrophobic membrane from GE Osmonics, Minnetonka, Minn.) thatdefines a wall of the channel. As described elsewhere herein,hydrophobic vents can be used to position a microdroplet of sample at adesired location within a microfluidic network.

The hydrophobic vents of the present technology are preferablyconstructed so that the amount of air that escapes through them ismaximized while minimizing the volume of the channel below the ventsurface. Accordingly, it is preferable that the vent is constructed soas to have a hydrophobic membrane 1303 of large surface area and ashallow cross section of the microchannel below the vent surface.

Hydrophobic vents are useful for bubble removal and typically have alength of at least about 2.5 mm (e.g., at least about 5 mm, at leastabout 7.5 mm) along a channel 1305 (see FIG. 13). The length of thehydrophobic vent is typically at least about 5 times (e.g., at leastabout 10 times, at least about 20 times) larger than a depth of thechannel within the hydrophobic vent. For example, in some embodiments,the channel depth within the hydrophobic vent is about 300 microns orless (e.g., about 250 microns or less, about 200 microns or less, about150 microns or less).

The depth of the channel within the hydrophobic vent is typically about75% or less (e.g., about 65% or less, about 60% or less) of the depth ofthe channel upstream 1301 and downstream (not shown) of the hydrophobicvent. For example, in some embodiments the channel depth within thehydrophobic vent is about 150 microns and the channel depth upstream anddownstream of the hydrophobic vent is about 250 microns. Otherdimensions are consistent with the description herein.

A width of the channel within the hydrophobic vent is typically at leastabout 25% wider (e.g., at least about 50% wider) than a width of thechannel upstream from the vent and downstream from the vent. Forexample, in an exemplary embodiment, the width of the channel within thehydrophobic vent is about 400 microns, and the width of the channelupstream and downstream from the vent is about 250 microns. Otherdimensions are consistent with the description herein.

The vent in FIG. 13 is shown in a linear configuration though it wouldbe understood that it need not be so. A bent, kinked, curved, S-shaped,V-shaped, or U-shaped (as in item 208 FIG. 11) vent is also consistentwith the manner of construction and operation described herein.

Heater Configurations to Ensure Uniform Heating of a Region

The microfluidic cartridges described herein are configured to positionin a complementary receiving bay in an apparatus that contains a heaterunit. The heater unit is configured to deliver heat to specific regionsof the cartridge, including but not limited to one or more microfluidiccomponents, at specific times. For example, the heat source isconfigured so that particular heating elements are situated adjacent tospecific components of the microfluidic network of the cartridge. Incertain embodiments, the apparatus uniformly controls the heating of aregion of a microfluidic network. In an exemplary embodiment, multipleheaters can be configured to simultaneously and uniformly heat a singleregion, such as the PCR reaction chamber, of the microfluidic cartridge.In other embodiments, portions of different sample lanes are heatedsimultaneously and independently of one another.

FIG. 14 shows a cross-sectional view of an exemplary microfluidiccartridge to show the location of a PCR channel in relation to variousheaters when the cartridge is placed in a suitable apparatus. The viewin FIG. 14 is also referred to as a sectional-isometric view of thecartridge lying over a heater wafer. A window 903 above the PCR channelin the cartridge is shown in perspective view. PCR channel 901 (forexample, 150μ deep×700μ wide), is shown in an upper layer of thecartridge. A laminate layer 905 of the cartridge (for example, 125μthick) is directly under the PCR channel 901. As depicted, an optionalfurther layer of thermal interface laminate 907 on the cartridge (forexample, 125μ thick) lies directly under the laminate layer 905. Heaters909, 911 are situated in a heater substrate layer 913 directly under thethermal interface laminate, shown in cross-section. In one embodimentthe heaters are photolithographically defined and etched metal layers ofgold (typically about 3,000 Å thick). Layers of 400 Å of TiW (not shown)are deposited on top and bottom of the gold layer to serve as anadhesion layer. The substrate 913 used is glass, fused silica or aquartz wafer having a thickness of 0.4 mm, 0.5 mm, 0.7 mm, or 1 mm. Athin electrically-insulative layer of 2 μm silicon oxide serves as aninsulative layer on top of the metal layer. Additional thin electricallyinsulative layers such as 2-4 μm of Parylene may also be deposited ontop of the silicon oxide surface. Two long heaters 909 and 911, asfurther described herein, run alongside the PCR channel.

An exemplary heater array is shown in FIGS. 15A and 15B. Additionalembodiments of heater arrays are described in U.S. patent applicationSer. No. 11/940,315, entitled “Heater Unit for Microfluidic DiagnosticSystem” and filed on even date herewith, the specification of which isincorporated herein by reference in its entirety.

Referring to FIGS. 15A and 15B, an exemplary PCR reaction chamber 1501,typically having a volume ˜1.6 μl, is configured with a long side and ashort side, each with an associated heating element. The heatersubstrate therefore includes four heaters disposed along the sides of,and configured to heat, the PCR reaction chamber, as shown in theexemplary embodiment of FIG. 15A: long top heater 1505, long bottomheater 1503, short left heater 1507, and short right heater 1509. Thesmall gap between long top heater 1505 and long bottom heater 1503results in a negligible temperature gradient (less than 1° C. differenceacross the width of the PCR channel at any point along the length of thePCR reaction chamber) and therefore an effectively uniform temperaturethroughout the PCR reaction chamber. The heaters on the short edges ofthe PCR reactor provide heat to counteract the gradient created by thetwo long heaters from the center of the reactor to the edge of thereactor. It would be understood by one of ordinary skill in the art thatstill other configurations of one or more heater(s) situated about a PCRreaction chamber are consistent with the methods and apparatus describedherein. For example, a ‘long’ side of the reaction chamber can beconfigured to be heated by two or more heaters. Specific orientationsand configurations of heaters are used to create uniform zones ofheating even on substrates having poor thermal conductivity because thepoor thermal conductivity of glass, or quartz, polyimide, FR4, ceramic,or fused silica substrates is utilized to help in the independentoperation of various microfluidic components such as valves andindependent operation of the various PCR lanes. In FIG. 15B, variousaspects of fine structure of heater elements are shown in inserts.

Generally, the heating of microfluidic components, such as a PCRreaction chamber, is controlled by passing currents through suitablyconfigured microfabricated heaters. Under control of suitable circuitry,the lanes of a multi-lane cartridge can then be controlled independentlyof one another. This can lead to a greater energy efficiency of theapparatus, because not all heaters are heating at the same time, and agiven heater is receiving current for only that fraction of the timewhen it is required to heat. Control systems and methods of controllablyheating various heating elements are further described in U.S. patentapplication Ser. No. 11/940,315, filed Nov. 14, 2007 and entitled“Heater Unit for Microfluidic Diagnostic System”.

The configuration for uniform heating, shown in FIG. 15A for a singlePCR reaction chamber, can be applied to a multi-lane PCR cartridge inwhich multiple independent PCR reactions occur. See, e.g., FIG. 15C,which shows an array of heater elements suitable to heat the cartridgeof FIG. 1.

In other embodiments, as further described in U.S. patent applicationSer. No. 11/940,315, filed Nov. 14, 2007 and entitled “Heater Unit forMicrofluidic Diagnostic System”, the heaters may have an associatedtemperature sensor, or may themselves function as sensors.

Use of Cutaways in Cartridge and Substrate to Improve Rate of CoolingDuring PCR Cycling

During a PCR amplification of a nucleotide sample, a number of thermalcycles are carried out. For improved efficiency, the cooling betweeneach application of heat is preferably as rapid as possible. Improvedrate of cooling can be achieved with various modifications to theheating substrate and/or the cartridge, as shown in FIG. 16.

One way to achieve rapid cooling is to cutaway portions of themicrofluidic cartridge substrate, as shown in FIG. 16. The upper panelof FIG. 16 is a cross-section of an exemplary microfluidic cartridgetaken along the dashed line A-A′ as marked on the lower panel of FIG.16. PCR reaction chamber 1601, and representative heaters 1603 areshown. Also shown are two cutaway portions, one of which labeled 1601,that are situated alongside the heaters that are positioned along thelong side of the PCR reaction chamber. Cutaway portions such as 1601reduce the thermal mass of the cartridge, and also permit air tocirculate within the cutaway portions. Both of these aspects permit heatto be conducted away quickly from the immediate vicinity of the PCRreaction chamber. Other configurations of cutouts, such as in shape,position, and number, are consistent with the present technology.

Another way to achieve rapid cooling is to cutaway portions of theheater substrate, and also to use ambient air cooling, as furtherdescribed in U.S. patent application Ser. No. 11/940,315, filed Nov. 14,2007 and entitled “Heater Unit for Microfluidic Diagnostic System”.

An example of thermal cycling performance in a PCR reaction chamberobtained with a configuration as described herein, is shown in FIG. 17for a protocol that is set to heat up the reaction mixture to 92° C.,and maintain the temperature for 1 second, then cool to 62° C., and stayfor 10 seconds. The cycle time shown is about 29 seconds, with 8 secondsrequired to heat from 62° C. and stabilize at 92° C., and 10 secondsrequired to cool from 92° C., and stabilize at 62° C. To minimize theoverall time required for a PCR effective to produce detectablequantities of amplified material, it is important to minimize the timerequired for each cycle. Cycle times in the range 15-30 s, such as 18-25s, and 20-22 s, are desirable. In general, an average PCR cycle time of25 seconds as well as cycle times as low as 20 seconds are typical withthe technology described herein. Using reaction volumes less than amicroliter (such as a few hundred nanoliters or less) permits use of anassociated smaller PCR chamber, and enables cycle times as low as 15seconds.

Manufacturing Process for Cartridge

FIG. 18 shows a flow-chart 1800 for an embodiment of an assembly processfor an exemplary cartridge as shown in FIG. 4A herein. It would beunderstood by one of ordinary skill in the art, both that various stepsmay be performed in a different order from the order set forth in FIG.18, and additionally that any given step may be carried out byalternative methods to those described in the figure. It would also beunderstood that, where separate serial steps are illustrated forcarrying out two or more functions, such functions may be performedsynchronously and combined into single steps and remain consistent withthe overall process described herein.

At 1802, a laminate layer is applied to a microfluidic substrate thathas previously been engineered, for example by injection molding, tohave a microfluidic network constructed in it; edges are trimmed fromthe laminate where they spill over the bounds of the substrate.

At 1804, wax is dispensed and loaded into the microvalves of themicrofluidic network in the microfluidic substrate. An exemplary processfor carrying this out is further described herein.

At 1806, the substrate is inspected to ensure that wax from step 1804 isloaded properly and that the laminate from step 1802 adheres properly toit. If a substrate does not satisfy either or both of these tests, it isusually discarded. If substrates repeatedly fail either or both of thesetests, then the wax dispensing, or laminate application steps, asapplicable, are reviewed.

At 1808, a hydrophobic vent membrane is applied to, and heat bonded to,the top of the microfluidic substrate covering at least the one or morevent holes, and on the opposite face of the substrate from the laminate.Edges of the membrane that are in excess of the boundary of thesubstrate are trimmed.

At 1810, the assembly is inspected to ensure that the hydrophobic ventmembrane is bonded well to the microfluidic substrate withoutheat-clogging the microfluidic channels. If any of the channels isblocked, or if the bond between the membrane and the substrate isimperfect, the assembly is discarded, and, in the case of repeateddiscard events, the foregoing process step 1808 is reviewed.

At 1812, optionally, a thermally conductive pad layer is applied to thebottom laminate of the cartridge.

At 1814, two label strips are applied to the top of the microfluidicsubstrate, one to cover the valves, and a second to protect the ventmembranes. It would be understood that a single label strip may bedevised to fulfill both of these roles.

At 1816, additional labels are printed or applied to show identifyingcharacteristics, such as a barcode #, lot # and expiry date on thecartridge. Preferably one or more of these labels has a space and awritable surface that permits a user to make an identifying annotationon the label, by hand.

Optionally, at 1818, to facilitate transport and delivery to a customer,assembled and labeled cartridges are stacked, and cartridges packed intogroups, such as groups of 25, or groups of 10, or groups of 20, orgroups of 48 or 50. Preferably the packaging is via an inert and/ormoisture-free medium.

Wax Loading in Valves

In general, a valve as shown in, e.g., FIGS. 12A-C, is constructed bydepositing a precisely controlled amount of a TRS (such as wax) into aloading inlet machined in the microfluidic substrate. FIGS. 19A and 19Bshow how a combination of controlled hot drop dispensing into a heatedmicrochannel device of the right dimensions and geometry is used toaccurately load wax into a microchannel of a microfluidic cartridge toform a valve. The top of FIG. 19A shows a plan view of a valve inlet1901 and loading channel 1902, connecting to a flow channel 1904. Thelower portions of FIG. 19A show the progression of a dispensed waxdroplet 1906 (having a volume of 75 nl+15 nl) through the inlet 1901 andinto the loading channel 1902.

To accomplish those steps, a heated dispenser head can be accuratelypositioned over the inlet hole of the microchannel in the microfluidicdevice, and can dispense molten wax drops in volumes as small as 75nanoliters with an accuracy of 20%. A suitable dispenser is also onethat can deposit amounts smaller than 100 nl with a precision of +/−20%.The dispenser should also be capable of heating and maintaining thedispensing temperature of the TRS to be dispensed. For example, it mayhave a reservoir to hold the solution of TRS. It is also desirable thatthe dispense head can have freedom of movement at least in a horizontal(x−y) plane so that it can easily move to various locations of amicrofluidic substrate and dispense volumes of TRS into valve inlets atsuch locations without having to be re-set, repositioned manually, orrecalibrated in between each dispense operation.

The inlet hole of the microfluidic cartridge, or other microchanneldevice, is dimensioned in such a way that the droplet of 75 nl can beaccurately propelled to the bottom of the inlet hole using, for example,compressed air, or in a manner similar to an inkjet printing method. Themicrofluidic cartridge is maintained at a temperature above the meltingpoint of the wax thereby permitting the wax to stay in a molten stateimmediately after it is dispensed. After the drop falls to the bottom ofthe inlet hole 1901, the molten wax is drawn into the narrow channel bycapillary action, as shown in the sequence of views in FIG. 19B. Ashoulder between the inlet hole 1901 and the loading channel canfacilitate motion of the TRS. The volume of the narrow section can bedesigned to be approximately equal to a maximum typical amount that isdispensed into the inlet hole. The narrow section can also be designedso that even though the wax dispensed may vary considerably between aminimum and a maximum shot size, the wax always fills up to, and stopsat, the microchannel junction 1907 because the T-junction provides ahigher cross section than that of the narrow section and thus reducesthe capillary forces. Dimensions shown in FIG. 19A are exemplary.

PCR Reagent Mixtures

In various embodiments, the sample for introduction into a lane of themicrofluidic cartridge can include a PCR reagent mixture comprising apolymerase enzyme and a plurality of nucleotides.

In various embodiments, preparation of a PCR-ready sample for use withan apparatus and cartridge as described herein can include contacting aneutralized polynucleotide sample with a PCR reagent mixture comprisinga polymerase enzyme and a plurality of nucleotides (in some embodiments,the PCR reagent mixture can further include a positive control plasmidand a fluorogenic hybridization probe selective for at least a portionof the plasmid).

The PCR-ready sample can be prepared, for example, using the followingsteps: (1) collect sample in sample collection buffer, (2) transferentire sample to lysis tube, mix, heat, and incubate for seven minutes,(3) place on magnetic rack, allow beads to separate, aspiratesupernatant, (4) add 100 μl of Buffer 1, mix, place on magnetic rack,allow beads to separate, aspirate supernatant, (5) add 10 A1 of Buffer2, mix, place in high temperature heat block for 3 minutes, place onmagnetic rack, allow beads to separate, transfer 5 μl supernatant, and(6) Add 5 μl of Buffer 3, transfer 1 to 10 μl of supernatant for PCRamplification and detection.

The PCR reagent mixture can be in the form of one or more lyophilizedpellets and the steps by which the PCR-ready sample is prepared caninvolve reconstituting the PCR pellet by contacting it with liquid tocreate a PCR reagent mixture solution. In yet another embodiment, eachof the PCR lanes may have dried down or lyophilized ASR reagentspreloaded such that the user only needs to input prepared polynucleotidesample into the PCR. In another embodiment, the PCR lanes may have onlythe application-specific probes and primers pre-measured and pre-loaded,and the user inputs a sample mixed with the PCR reagents.

In various embodiments, the PCR-ready sample can include at least oneprobe that can be selective for a polynucleotide sequence, wherein thesteps by which the PCR-ready sample is prepared involve contacting theneutralized polynucleotide sample or a PCR amplicon thereof with theprobe. The probe can be a fluorogenic hybridization probe. Thefluorogenic hybridization probe can include a polynucleotide sequencecoupled to a fluorescent reporter dye and a fluorescence quencher dye.

In various embodiments, the PCR-ready sample further includes a samplebuffer.

In various embodiments, the PCR-ready sample includes at least one probethat is selective for a polynucleotide sequence, e.g., thepolynucleotide sequence that is characteristic of a pathogen selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the PCR reagent mixture can further include apolymerase enzyme, a positive control plasmid and a fluorogenichybridization probe selective for at least a portion of the plasmid.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism, for example any organismthat employs deoxyribonucleic acid or ribonucleic acid polynucleotides.Thus, the probe can be selective for any organism. Suitable organismsinclude mammals (including humans), birds, reptiles, amphibians, fish,domesticated animals, wild animals, extinct organisms, bacteria, fungi,viruses, plants, and the like. The probe can also be selective forcomponents of organisms that employ their own polynucleotides, forexample mitochondria. In some embodiments, the probe is selective formicroorganisms, for example, organisms used in food production (forexample, yeasts employed in fermented products, molds or bacteriaemployed in cheeses, and the like) or pathogens (e.g., of humans,domesticated or wild mammals, domesticated or wild birds, and the like).In some embodiments, the probe is selective for organisms selected fromthe group consisting of gram positive bacteria, gram negative bacteria,yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism selected from the groupconsisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus,Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistantStaphylococcus; Streptococcus(e.g., α, β or γ-hemolytic, Group A, B, C,D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, andE. faecium (formerly S. faecalis, S. durans, S. faecium);nonenterococcal group D streptococci, e.g., S. bovis and S. equines;Streptococcus viridans, e.g., S. mutans, S. sanguis, S. salivarius, S.mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus;S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides, N.gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E.rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanoviiand L. seeligeri; Bacillus, e.g., B. anthracis, B. cereus, B. subtilis,B. subtilis niger, B. thuringiensis; Nocardia asteroids; Legionella,e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii;Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E.coli, E. coliO157:H7); Klebsiella, Enterobacter, Serratia, Proteus,Morganella, Providencia, Yersinia, and the like, e.g., Salmonella, e.g.,S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii),S. dublin S. choleraesuis, S. enteritidis, S. typhimurium, S.heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S.saint-paul; Shigella e.g., subgroups: A, B, C, and D, such as S.flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis,P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia(P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y.pestis, Y. enterocolitica; Haemophilus, e.g., H. influenzae, H.parainfluenzae H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus,B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis;Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei,Burkholderia mallei, Burkholderia cepacia and Stenotrophomonasmaltophilia; Campylobacter, e.g., C. fetus fetus, C. jejuni, C. pylori(Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus,V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and thenonagglutinable vibrios; Clostridia, e.g., C. perfringens, C. tetani, C.difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides,e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B.ovatus, B. caccae, and B. merdae; Prevotella, e.g., P. melaninogenica;genus Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum,T. pallidum subspecies pertenue, T. carateum, and T. pallidum subspeciespallidum; genus Borrelia, e.g., B burgdorferi; genus Leptospira;Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus;Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. aviumM. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, theM. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M.asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M.malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma,e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae,M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M.genitalium; Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T.vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H.capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides,e.g., C. immitis; Blastomyces, e.g. B. dermatitidis; Paracoccidioides,e.g., P. brasiliensis; Penicillium, e.g., P. marneffei; Sporothrix,e.g., S. schenckii; Rhizopus, Rhizomucor, Absidia, and Basidiobolus;diseases caused by Bipolaris, Cladophialophora, Cladosporium,Drechslera, Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis,Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T.beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P.falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa ofthe genus Trypanosoma, e.g., T. cruzi; Leishmania, e.g., L. donovani, L.major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis;Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria orAcanthamoeba; Entamoeba histolytica; Giardia lamblia; genusCryptosporidium, e.g., C. parvum; Isospora belli; Cyclosporacayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostomaduodenale or Necator americanus; Strongyloides stercoralis Toxocara,e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella,e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocercavolvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S.japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium;Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis;Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothriumlatum; Taenia, e.g., T. saginata, T. solium; Echinococcus, e.g., E.granulosus, E. multilocularis; Picornaviruses, rhinoviruses echoviruses,coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3,and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;varicella-zoster virus; human T-lymphotrophic virus (type I and typeII); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae,Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis(alphaviruses [e.g., Venezuelan equine encephalitis, eastern equineencephalitis, western equine encephalitis]); Viral hemorrhagic fevers(filoviruses [e.g., Ebola, Marburg] and arenaviruses [e.g., Lassa,Machupo]); Smallpox (variola); retroviruses e.g., human immunodeficiencyviruses 1 and 2; human papillomavirus [HPV] types 6, 11, 16, 18, 31, 33,and 35.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organisms selected from the groupconsisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiellaoxytoca, Klebsiella pneumoniae, Escherichia coli, AcinetobacterBaumannii, Serratia marcescens, Enterobacter aerogenes, Enterococcusfaecium, vancomycin-resistant enterococcus (VRE), Staphylococcus aureus,methicillin-resistant Staphylococcus aureus(MRSA), Streptococcusviridans, Listeria monocytogenes, Enterococcus spp., Streptococcus GroupB, Streptococcus Group C, Streptococcus Group G, Streptococcus Group F,Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcusepidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilusinfluenzae, Neisseria gonorrhoeee, Moraxella catarrahlis, Salmonellasps., Chlamydia trachomatis, Peptostreptococcus productus,Peptostreptococcus anaerobius, Lactobacillus fermentum, Eubacteriumlentum, Candida glabrata, Candida albicans, Chlamydia spp.,Campylobacter spp., Salmonella spp., smallpox (variola major), YersiniaPestis, Herpes Simplex Virus I (HSV I), and Herpes Simplex Virus II (HSVII).

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of Group B Streptococcus.

In various embodiments, a method of carrying out PCR on a sample canfurther include one or more of the following steps: heating thebiological sample in the microfluidic cartridge; pressurizing thebiological sample in the microfluidic cartridge at a pressuredifferential compared to ambient pressure of between about 20kilopascals and 200 kilopascals, or in some embodiments, between about70 kilopascals and 110 kilopascals.

In some embodiments, the method for sampling a polynucleotide caninclude the steps of: placing a microfluidic cartridge containing aPCR-ready sample in a receiving bay of a suitably configured apparatus;carrying out PCR on the sample under thermal cycling conditions suitablefor creating PCR amplicons from the neutralized polynucleotide in thesample, the PCR-ready sample comprising a polymerase enzyme, a positivecontrol plasmid, a fluorogenic hybridization probe selective for atleast a portion of the plasmid, and a plurality of nucleotides;contacting the neutralized polynucleotide sample or a PCR ampliconthereof with the at least one fluorogenic probe that is selective for apolynucleotide sequence, wherein the probe is selective for apolynucleotide sequence that is characteristic of an organism selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses; and detecting thefluorogenic probe, the presence of the organism for which the onefluorogenic probe is selective is determined.

Carrying out PCR on a PCR-ready sample can additionally include:independently contacting each of the neutralized polynucleotide sampleand a negative control polynucleotide with the PCR reagent mixture underthermal cycling conditions suitable for independently creating PCRamplicons of the neutralized polynucleotide sample and PCR amplicons ofthe negative control polynucleotide; and/or contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof and the negative controlpolynucleotide or a PCR amplicon thereof with at least one probe that isselective for a polynucleotide sequence.

In various embodiments, a method of using the apparatus and cartridgedescribed herein can further include one or more of the following steps:determining the presence of a polynucleotide sequence in the biologicalsample, the polynucleotide sequence corresponding to the probe, if theprobe is detected in the neutralized polynucleotide sample or a PCRamplicon thereof; determining that the sample was contaminated if theprobe is detected in the negative control polynucleotide or a PCRamplicon thereof; and/or in some embodiments, wherein the PCR reagentmixture further comprises a positive control plasmid and a plasmid probeselective for at least a portion of the plasmid, the method furtherincluding determining that a PCR amplification has occurred if theplasmid probe is detected.

Kit

In various embodiments, the microfluidic cartridge as described hereincan be provided in the form of a kit, wherein the kit can include amicrofluidic cartridge, and a liquid transfer member (such as a syringeor a pipette). In various embodiments, the kit can further includeinstructions to employ the liquid transfer member to transfer a samplecontaining extracted nucleic acid from a sample container via a sampleinlet to the microfluidic network on the microfluidic cartridge. In someembodiments, the microfluidic cartridge and the liquid transfer membercan be sealed in a pouch with an inert gas.

Typically when transferring a sample from liquid dispenser, such as apipette tip, to an inlet on the microfluidic cartridge, a volume of airis simultaneously introduced into the microfluidic network, the volumeof air being between about 0.5 mL and about 5 mL. Presence of air in themicrofluidic network, however, is not essential to operation of thecartridge described herein.

In various embodiments, the kit can further include at least onecomputer-readable label on the cartridge. The label can include, forexample, a bar code, a radio frequency tag or one or morecomputer-readable characters. When used in conjunction with a similarcomputer-readable label on a sample container, such as a vial or apouch, matching of diagnostic results with sample is therebyfacilitated.

In some embodiments, a sample identifier of the apparatus describedelsewhere herein is employed to read a label on the microfluidiccartridge and/or a label on the biological sample.

Overview of an Apparatus for Receiving a Microfluidic Cartridge

The present technology relates to a cartridge, complementary apparatus,and related methods for amplifying, and carrying out diagnostic analyseson, nucleotides from biological samples. The technology includes adisposable or reusable microfluidic cartridge containing multiple samplelanes capable of processing samples in parallel as described elsewhereherein, and a reusable apparatus that is configured to selectivelyactuate on-cartridge operations, to detect and analyze the products ofthe PCR amplification in each of the lanes separately, in allsimultaneously, or in groups simultaneously, and, optionally, candisplay the progression of analyses and results thereof on a graphicaluser interface. Such a reusable apparatus is further described in U.S.patent application Ser. No. 11/985,577, entitled “Microfluidic Systemfor Amplifying and Detecting Polynucleotides in Parallel” and filed onNov. 14, 2007, and which is incorporated herein by reference in itsentirety.

FIG. 20 shows a perspective view of an exemplary apparatus 2000consistent with those described herein, as well as various componentsthereof, such as exemplary cartridge 2010 that contains multiple samplelanes, and exemplary read head 2020 that contains detection apparatusfor reading signals from cartridge 2010. The apparatus 2000 of FIG. 20is able to carry out real-time PCR on a number of samples in cartridge2010 simultaneously or serially. Preferably the number of samples is 12samples, as illustrated with exemplary cartridge 2010, though othernumbers of samples such as 4, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and48 are within the scope of the present description. In preferredoperation of the apparatus, a PCR-ready solution containing the sample,and, optionally, one or more analyte-specific reagents (ASR's) isprepared, as further described elsewhere (see, e.g., U.S. patentapplication publication 2006-0166233, incorporated herein by reference),prior to introduction into cartridge 200.

In some embodiments, an apparatus includes: a receiving bay configuredto selectively receive a microfluidic cartridge as described herein; atleast one heat source thermally coupled to the receiving bay; and aprocessor coupled to the heat source, wherein the heat source isconfigured to selectively heat individual regions of individual samplelanes in the cartridge, and the processor is configured to controlapplication of heat to the individual sample lanes, separately, in allsimultaneously, or in groups simultaneously; at least one detectorconfigured to detect one or more polynucleotides or a probe thereof in asample in one or more of the individual sample lanes, separately orsimultaneously; and a processor coupled to the detector to control thedetector and to receive signals from the detector.

FIG. 21 shows a schematic cross-sectional view of a part of an apparatusas described herein, showing input of sample into a cartridge 2100 via apipette 10 (such as a disposable pipette) and an inlet 202. Cartridge2100 is situated in a suitably configured receiving bay 2112. Inlet 2102is preferably configured to receive a pipette or the bottom end of a PCRtube and thereby accept sample for analysis with minimum waste, and withminimum introduction of air. Cartridge 2100 is disposed on top of and incontact with a heater substrate 2140. Read head 2130 is positioned abovecartridge 2100 and a cover for optics 2131 restricts the amount ofambient light that can be detected by the read head.

FIG. 22 shows an example of 4-pipette head used for attaching disposablepipette tips, prior to dispensing PCR-ready sample into a cartridge asfurther described herein.

The receiving bay is a portion of the apparatus that is configured toselectively receive the microfluidic cartridge. For example, thereceiving bay and the microfluidic cartridge can be complementary inshape so that the microfluidic cartridge is selectively received in,e.g., a single orientation. The microfluidic cartridge can have aregistration member that fits into a complementary feature of thereceiving bay. The registration member can be, for example, a cut-out onan edge of the cartridge, such as a corner that is cut-off, or one ormore notches or grooves that are made on one or more of the sides in adistinctive pattern that prevents a cartridge from being loaded into thebay in more than one distinct orientation. By selectively receiving thecartridge, the receiving bay can help a user to place the cartridge sothat the apparatus can properly operate on the cartridge. The cartridgecan be designed to be slightly smaller than the dimensions of thereceiving bay, for example by approximately 200-300 microns, for easyplacement and removal of the cartridge.

The receiving bay can also be configured so that various components ofthe apparatus that operate on the microfluidic cartridge (heat sources,detectors, force members, and the like) are positioned to properlyoperate thereon. For example, a contact heat source can be positioned inthe receiving bay such that it can be thermally coupled to one or moredistinct locations on a microfluidic cartridge that is selectivelyreceived in the bay. Microheaters in the heater module as furtherdescribed elsewhere herein were aligned with correspondingheat-requiring microcomponents (such as valves, pumps, gates, reactionchambers, etc). The microheaters can be designed to be slightly biggerthan the heat requiring microfluidic components so that even though thecartridge may be off-centered from the heater, the individual componentscan still function effectively.

As further described elsewhere herein, the lower surface of thecartridge can have a layer of mechanically compliant heat transferlaminate that can enable thermal contact between the microfluidicsubstrate and the microheater substrate of the heater module. A minimalpressure of 1 psi can be employed for reliable operation of the thermalvalves, gates and pumps present in the microfluidic cartridge.

In various embodiments of the apparatus, the apparatus can furtherinclude a sensor coupled to the processor, the sensor configured tosense whether the microfluidic cartridge is selectively received.

The heat source can be, for example, a heat source such as a resistiveheater or network of resistive heaters. In preferred embodiments, the atleast one heat source can be a contact heat source selected from aresistive heater (or network thereof), a radiator, a fluidic heatexchanger and a Peltier device. The contact heat source can beconfigured at the receiving bay to be thermally coupled to one or moredistinct locations of a microfluidic cartridge received in the receivingbay, whereby the distinct locations are selectively heated. The contactheat source typically includes a plurality of contact heat sources, eachconfigured at the receiving bay to be independently thermally coupled toa different distinct location in a microfluidic cartridge receivedtherein, whereby the distinct locations are independently heated. Thecontact heat sources can be configured to be in direct physical contactwith one or more distinct locations of a microfluidic cartridge receivedin the bay. In various embodiments, each contact source heater can beconfigured to heat a distinct location having an average diameter in 2dimensions from about 1 millimeter (mm) to about 15 mm (typically about1 mm to about 10 mm), or a distinct location having a surface area ofbetween about 1 mm² about 225 mm² (typically between about 1 mm² andabout 100 mm², or in some embodiments between about 5 mm² and about 50mm²). Various configurations of heat sources are further described inU.S. patent application Ser. No. 11/940,315, entitled “Heater Unit forMicrofluidic Diagnostic System” and filed on even date herewith.

In various embodiments, the heat source is disposed in a heating modulethat is configured to be removable from the apparatus.

In various embodiments, the apparatus can include a compliant layer atthe contact heat source configured to thermally couple the contact heatsource with at least a portion of a microfluidic cartridge received inthe receiving bay. The compliant layer can have a thickness of betweenabout 0.05 and about 2 millimeters and a Shore hardness of between about25 and about 100. Such a compliant layer may not be required if theinstrument is able to reliably press the cartridge over the heatersurface with a minimum contact pressure of say 1 psi over the entiretyof the cartridge.

The detector can be, for example, an optical detector. For example, thedetector can include a light source that selectively emits light in anabsorption band of a fluorescent dye, and a light detector thatselectively detects light in an emission band of the fluorescent dye,wherein the fluorescent dye corresponds to a fluorescent polynucleotideprobe or a fragment thereof. Alternatively, for example, the opticaldetector can include a bandpass-filtered diode that selectively emitslight in the absorption band of the fluorescent dye and a bandpassfiltered photodiode that selectively detects light in the emission bandof the fluorescent dye; or for example, the optical detector can beconfigured to independently detect a plurality of fluorescent dyeshaving different fluorescent emission spectra, wherein each fluorescentdye corresponds to a fluorescent polynucleotide probe or a fragmentthereof; or for example, the optical detector can be configured toindependently detect a plurality of fluorescent dyes at a plurality ofdifferent locations on a microfluidic cartridge, wherein eachfluorescent dye corresponds to a fluorescent polynucleotide probe or afragment thereof in a different sample. The detector can also beconfigured to detect the presence or absence of sample in a PCR reactionchamber in a given sample lane, and to condition initiation ofthermocycling upon affirmative detection of presence of the sample.Further description of suitably configured detectors are described inU.S. patent application Ser. No. 11/940,321, filed on Nov. 14, 2007 andentitled “Fluorescence Detector for Microfluidic Diagnostic System”,incorporated herein by reference.

Although the various depictions therein show a heater substrate disposedunderneath a microfluidic substrate, and a detector disposed on top ofit, it would be understood that an inverted arrangement would workequally as well. In such an embodiment, the heater would be forced downonto the microfluidic substrate, making contact therewith, and thedetector would be mounted underneath the substrate, disposed to collectlight directed downwards towards it.

In another preferred embodiment (not shown in the FIGs. herein), acartridge and apparatus are configured so that the read-head does notcover the sample inlets, thereby permitting loading of separate sampleswhile other samples are undergoing PCR thermocycling.

In various embodiments, the apparatus can further include an analysisport. The analysis port can be configured to allow an external sampleanalyzer to analyze a sample in the microfluidic cartridge. For example,the analysis port can be a hole or window in the apparatus which canaccept an optical detection probe that can analyze a sample or progressof PCR in situ in the microfluidic cartridge. In some embodiments, theanalysis port can be configured to direct a sample from the microfluidiccartridge to an external sample analyzer; for example, the analysis portcan include a conduit in fluid communication with the microfluidiccartridge that directs a liquid sample containing an amplifiedpolynucleotide to a chromatography apparatus, an optical spectrometer, amass spectrometer, or the like.

In various embodiments, the apparatus can further include one or moreforce members configured to apply force to at least a portion of amicrofluidic cartridge received in the receiving bay. The one or moreforce members are configured to apply force to thermally couple the atleast one heat source to at least a portion of the microfluidiccartridge. The application of force is important to ensure consistentthermal contact between the heater wafer and the PCR reactor andmicrovalves in the microfluidic cartridge.

The apparatus preferably also includes a processor, comprisingmicroprocessor circuitry, in communication with, for example, the inputdevice and a display, that accepts a user's instructions and controlsanalysis of samples.

In various embodiments, the apparatus can further include a lid at thereceiving bay, the lid being operable to at least partially excludeambient light from the receiving bay.

In various embodiments, the apparatus can further include at least oneinput device coupled to the processor, the input device being selectedfrom the group consisting of a keyboard, a touch-sensitive surface, amicrophone, and a mouse.

In various embodiments, the apparatus can further include at least onesample identifier coupled to the processor, the sample identifier beingselected from an optical scanner such as an optical character reader, abar code reader, or a radio frequency tag reader. For example, thesample identifier can be a handheld bar code reader.

In various embodiments, the apparatus can further include at least onedata storage medium coupled to the processor, the medium selected from:a hard disk drive, an optical disk drive, or one or more removablestorage media such as a CD-R, CD-RW, USB-drive, or flash memory card.

In various embodiments, the apparatus can further include at least oneoutput coupled to the processor, the output being selected from adisplay, a printer, and a speaker, the coupling being either directlythrough a directly dedicated printer cable, or wirelessly, or via anetwork connection.

The apparatus further optionally comprises a display that communicatesinformation to a user of the system. Such information includes but isnot limited to: the current status of the system; progress of PCRthermocycling; and a warning message in case of malfunction of eithersystem or cartridge. The display is preferably used in conjunction withan external input device as elsewhere described herein, through which auser may communicate instructions to apparatus 100. A suitable inputdevice may further comprise a reader of formatted electronic media, suchas, but not limited to, a flash memory card, memory stick, USB-stick,CD, or floppy diskette. An input device may further comprise a securityfeature such as a fingerprint reader, retinal scanner, magnetic stripreader, or barcode reader, for ensuring that a user of the system is infact authorized to do so, according to pre-loaded identifyingcharacteristics of authorized users. An input device mayadditionally—and simultaneously—function as an output device for writingdata in connection with sample analysis. For example, if an input deviceis a reader of formatted electronic media, it may also be a writer ofsuch media. Data that may be written to such media by such a deviceincludes, but is not limited to, environmental information, such astemperature or humidity, pertaining to an analysis, as well as adiagnostic result, and identifying data for the sample in question.

The apparatus may further include a computer network connection thatpermits extraction of data to a remote location, such as a personalcomputer, personal digital assistant, or network storage device such ascomputer server or disk farm. The network connection can be acommunications interface selected from the group consisting of: a serialconnection, a parallel connection, a wireless network connection, and awired network connection such as an ethernet or cable connection,wherein the communications interface is in communication with at leastthe processor. The computer network connection may utilize, e.g.,ethernet, firewire, or USB connectivity. The apparatus may further beconfigured to permit a user to e-mail results of an analysis directly tosome other party, such as a healthcare provider, or a diagnosticfacility, or a patient.

In various embodiments, there is an associated computer program productincludes computer readable instructions thereon for operating theapparatus and for accepting instructions from a user.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: output an indicator of theplacement of the microfluidic cartridge in the receiving bay; read asample label or a microfluidic cartridge label; output directions for auser to input a sample identifier; output directions for a user to loada sample transfer member with the PCR-ready sample; output directionsfor a user to introduce the PCR-ready sample into the microfluidiccartridge; output directions for a user to place the microfluidiccartridge in the receiving bay; output directions for a user to closethe lid to operate the force member; output directions for a user topressurize the PCR-ready sample in the microfluidic cartridge byinjecting the PCR-ready sample with a volume of air between about 0.5 mLand about 5 mL; and output status information for sample progress fromone or more lanes of the cartridge.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: heat the PCR ready-sampleunder thermal cycling conditions suitable for creating PCR ampliconsfrom the neutralized polynucleotide; contact the neutralizedpolynucleotide sample or a PCR amplicon thereof with at least one probethat is selective for a polynucleotide sequence; independently contacteach of the neutralized polynucleotide sample and a negative controlpolynucleotide with the PCR reagent mixture under thermal cyclingconditions suitable for independently creating PCR amplicons of theneutralized polynucleotide sample and PCR amplicons of the negativecontrol polynucleotide; contact the neutralized polynucleotide sample ora PCR amplicon thereof and the negative control polynucleotide or a PCRamplicon thereof with at least one probe that is selective for apolynucleotide sequence; output a determination of the presence of apolynucleotide sequence in the biological sample, the polynucleotidesequence corresponding to the probe, if the probe is detected in theneutralized polynucleotide sample or a PCR amplicon thereof; and/oroutput a determination of a contaminated result if the probe is detectedin the negative control polynucleotide or a PCR amplicon thereof.

Apparatus 100 may optionally comprise one or more stabilizing feet thatcause the body of the device to be elevated above a surface on whichsystem 100 is disposed, thereby permitting ventilation underneath system100, and also providing a user with an improved ability to lift system100.

EXAMPLES Example 1: 48 Lane Cartridge

FIG. 23 shows an exemplary 48-lane cartridge for carrying out PCRindependently on 48 samples, and with a reaction volume of 10 microlitereach. The area occupied by the entire cartridge is approximately 3.5inches (8.9 cm) by 4.25 inches (10.8 cm). The sample lanes are organizedas two groups of 24 each. The adjacent sample lanes in each of the tworows of 24 are spaced apart 4 mm (center-to-center). Trenches betweenthe PCR lanes may be cut in order to isolate the heating of each PCRchannel from those adjacent to it. This may be accomplished by etching,milling, controlled cutting, etc., during fabrication of the cartridge.

FIG. 24 shows a heater design used for actuating the 48 lane PCRcartridge of FIG. 23. The heating of each sample lane can beindependently controlled.

Example 2: PCR Cartridge with Post-PCR Retrieval Capability

Many applications such as genotyping, sequencing, multiple analytedetection (microarray, electrochemical sensing) require post-PCR sampleretrieval and subsequent analysis of the retrieved sample in a differentinstrument. The cartridge of this example, of which a 24 lane embodimentis shown in FIG. 25A, with a sample lane layout illustrated in FIG. 25B,accommodates such a retrieval capability. Each lane in the cartridge ofFIG. 25A independently permits sample retrieval. The configuration ofthe lane of FIG. 25B is different from that of, e.g., FIG. 6 at leastbecause of the presence of 2 gates and the alternative channel from thereactor, via Gate 1, to the inlet. Such features permit effective sampleretrieval.

Sample DNA mixed with PCR enzymes is input into a sample lane 2501through the inlet hole 2502 in the microfluidic network described below.The valves 2506, 2504 (valves 1 and 2) are initially open while thegates 2522, 2520 (gates 1 and 2) are closed, enabling the reaction mixto fill up the PCR reactor 2510 with the excess air venting out throughvent hole 1 (label 2514). The valves 1 and 2 are then closed to seal offthe reaction mixture. Thermocycling is initiated to conduct the PCRreaction within the PCR reactor. After the reaction is completed, apipette is mechanically interfaced with the inlet hole 2502 and suctionforce applied to the pipette. Gates 1 and 2 are opened to enable thereacted sample to exit the PCR reactor and enter the pipette. Thiscontrolled opening of the PCR device will also prevent post-PCRcontamination of the apparatus in which the cartridge resides as thereis minimal exposure of the PCR product with the atmosphere.

It will be understood that reactions other than PCR can easily beperformed in the cartridge of this example.

Example 3: 12-Lane Cartridge

The 12 channel cartridge of this example is the same basic design thatis described and shown in FIG. 3, with the following modifications: thevolume of the PCR reactor is increased from 2 μl to 4.5 μl, leading toan increase in the acceptable input volume from 4 μl to 6 μl. Increasingthe reaction volume facilitates detection from even dilute samples(wherein the target DNA concentration may be low). In order to detectDNA in a reactor of say 1 microliter volume, there should be a minimumof 1-2 copies of the DNA in the 1 microliter for positiveidentification, i.e., the concentration should not be less than around1-2 copies/microliter. Increasing the reaction volume to say 5microliters will reduce the minimum acceptable starting DNAconcentration by 5 fold. The inlet holes are moved a few millimetersaway from the edge of the cartridge to allow room for a 2 mm alignmentledge in the cartridge. A similar alignment ledge is also included onthe other edge of the cartridge. The alignment ledge permits thecartridges to be stacked during storage (or within a multi-cartridgespring-loader) without the hydrophobic vent of one cartridge coming intocontact with a surface of an adjacent cartridge.

Example 4: 24-Lane Cartridge

This 24-lane cartridge has two rows of 12 sample lanes. Each lane has: aliquid inlet port, that interfaces with a disposable pipette; a 4microliter PCR reaction chamber (1.5 mm wide, 300 microns deep andapproximately 10 mm long), and two microvalves on either side of the PCRreactor and outlet vent. Microvalves are normally open, and close thechannel on actuation. The outlet holes enable extra liquid (˜1 μl) to becontained in the fluidic channel in case more than 6 μl of fluid isdispensed into the cartridge. Thus, the cartridge of this example doesnot require a bubble vent as it will be used in an automated PCR machinehaving a reliable, precision liquid dispenser.

The inlet holes of the cartridge of this example are made conical inshape and have a diameter of 3-6 mm at the top to ensure that pipettetips can be easily landed by an automated fluid dispensing head into theconical hole, with some tolerance. There is also an optional raisedannulus around the top of the holes. Once the pipette tip lands withinthe cone, the conical shape guides the pipette and mechanically seals toprovide error free dispensing into, or withdrawal of fluid from, thecartridge. The bigger the holes, the better it is to align with thepipette, however, given the opposing need to maximize the number ofinlet ports within the width of the cartridge as well as to maintain thepitch between holes compatible with the inter-pipette distance, theholes cannot be too big. In this design, the inter-pipette tip distanceis 18 mm and the distance between the loading holes in the cartridge is6 mm. So lanes 1, 4, 7, 11 are pipetted into during one dispensingoperation that utilizes four pipette tips; lanes 2, 5, 8 and 12 in thenext, and so on and so forth.

The height of the conical holes is kept lower than the height of theledges on the edges of the cartridge to ensure the cartridges can bestacked on the ledges. The ledges on the two long edges of the cartridgeenable stacking of the cartridges with minimal surface contact betweentwo stacked cartridges and also help guide the cartridge into the readerfrom a spring-loader, where used.

Example 5: 12-Lane Cartridge

This 12-lane cartridge has 12 sample lanes in parallel, as shown inFIG. 1. Each lane has: a liquid inlet port that interfaces with adisposable pipette; a bubble vent; a PCR reaction chamber, and twomicrovalves on either side of the PCR reactor and outlet vent.Microvalves are normally open, and close the channel on actuation. Thereaction volume is in the range 1-10 μl so that the number of copies ofDNA will be sufficient for detection. Such a volume also permits the PCRreaction volume to be similar to release volume from a samplepreparation procedure.

Example 6: Kit

FIG. 26 shows a representative sample kit 2610 that includes amicrofluidic cartridge 2612 with a barcode label 2632, and one or moresample containers 2614 each also optionally having a barcode label.

FIG. 27 shows that one or more components of the sample kit, forexample, microfluidic cartridge 2612, can be packaged in a sealed pouch2624. The pouch can be hermetically sealed with an inert gas such asargon, nitrogen, or others.

The barcode labels of both cartridge and sample container can be readwith a bar code reader prior to use.

Example 7: Apparatus and Process for Wax Loading of Valves ExemplaryWax-Deposition Process

Deposition of wax in valves of the microfluidic network, as at step 1804of FIG. 18 may be carried out with the exemplary equipment shown inFIGS. 28A and 28B. The DispenseJet Series DJ-9000 (available fromAsymtek, Carlsbad, Calif.) is a non-contact dispenser suitable for thispurpose that provides rapid delivery and high-precision volumetriccontrol for various fluids, including surface mount adhesive, underfill,encapsulants, conformal coating, UV adhesives, and silver epoxy. TheDJ-9000 jets in tight spaces as small as 200 micrometers and createsfillet wet-out widths as small as 300 micrometers on the dispensed sideof a substrate such as a die. It dispenses fluid either as discrete dotsor a rapid succession of dots to form a 100-micron (4 mil) diameterstream of fluid from the nozzle. It is fully compatible with othercommercially available dispensing systems such as the Asymtek CenturyC-718/C-720, Millennium M-2000, and Axiom X-1000 Series DispensingSystems.

A DJ-9000 is manufactured under quality control standards that aim toprovide precise and reliable performance. Representative specificationsof the apparatus are as follows.

Characteristic Specification Size Width: 35 mm Height: 110 mm Depth: 100mm Weight 400 grams - dry Feed Tube Assembly Nylon - FittingPolyurethane - Tube Fluid Chamber Type 303 Stainless Steel Seat andNozzle 300/400 Series S/S, Carbide Needle Assembly 52100 Bearing Steel -Shaft Hard Chrome Plate Carbide - Tip Fluid Seal PEEK/Stainless SteelFluid Chamber 0-Ring Ethylene Propylene Jet Body 6061-T6 Aluminum NickelPlated Needle Assembly Bearings PEEK Thermal Control Body 6061-T6Aluminum Nickel Plated Reservoir Holder Acetyl Reservoir Size 5, 10, or30 cc (0.17, 0.34, or 1.0 oz) Feed Tube Assembly Fitting Female Luer perANSI/HIMA MD70.1-1983 Maximum Cycle Frequency 200 Hz. Minimum Valve AirPressure 5.5 bar (80 psi) Operating Noise Level 70 db* Solenoid 24 VDC,12.7 Watts Thermal Control Heater 24 VDC, 14.7 Watts, 40 ohms ThermalControl RTD 100 ohm, platinum Maximum Heater Set Point 80° C. *AtMaximum Cycle Rate

An exploded view of this apparatus is shown in FIG. 28B.

Theory of Operation of DJ-9000

The DJ-9000 has a normally closed, air-actuated, spring-returnmechanism, which uses momentum transfer principles to expel precisevolumes of material. Pressurized air is regulated by a high-speedsolenoid to retract a needle assembly from the seat. Fluid, fed into thefluid chamber, flows over the seat. When the air is exhausted, theneedle travels rapidly to the closed position, displacing fluid throughthe seat and nozzle in the form of a droplet. Multiple droplets fired insuccession can be used to form larger dispense volumes and lines whencombined with the motion of a dispenser robot.

The equipment has various adjustable features: The following featuresaffect performance of the DJ-9000 and are typically adjusted to fitspecific process conditions.

Fluid Pressure should be set so that fluid fills to the seat, but shouldnot be influential in pushing the fluid through the seat and nozzle. Ingeneral, higher fluid pressure results in a larger volume of materialjetted.

The Stroke Adjustment controls the travel distance of the NeedleAssembly. The control is turned counterclockwise to increase needleassembly travel, or turned clockwise to decrease travel. An increase oftravel distance will often result in a larger volume of material jetted.

The Solenoid Valve controls the valve operation. When energized, itallows air in the jet air chamber to compress a spring and thereby raisethe Needle Assembly. When de-energized, the air is released and thespring forces the piston down so that the needle tip contacts the seat.

The seat and nozzle geometry are typically the main factors controllingdispensed material volume. The seat and nozzle size are determined basedon the application and fluid properties. Other parameters are adjustedin accordance with seat and nozzle choices. Available seat and nozzlesizes are listed in the table hereinbelow.

Thermal Control Assembly: Fluid temperature often influences fluidviscosity and flow characteristics. The DJ-9000 is equipped with aThermal Control Assembly that assures a constant fluid temperature.

Dot and Line Parameters: In addition to the DJ-9000 hardwareconfiguration and settings, Dot and Line Parameters are set in asoftware program (referred to as FmNT) to control the size and qualityof dots and lines dispensed.

Example 7: 24-Lane Cartridge

FIGS. 29A-29C show an exemplary 24-lane cartridge having three layers inits construction in which there is no hydrophobic membrane, and nothermally compliant layer. The three layers are a laminate 2922, amicrofluidic substrate 2924, and a label 2926. A typical reaction vol.is 4.5 μl in each lane from 2 rows of 12 lanes. No bubble-removal ventsare utilized and instead of a hydrophobic end vent, there is just ahole. This is consistent with use of an accurate pipetting system. Thereis no thermally compliant/conductive layer for situations where enoughpressure can be reliably applied to the cartridge that effective thermalcontact with the microfluidic substrate can be made without requiringthe additional layer. The absence of two layers from the constructionsaves manufacturing costs.

Example 8: 96-Lane Cartridge

FIGS. 30A-D show aspects of a 96-lane cartridge design, includingcomplementary heater configurations. (FIG. 30A shows cartridge design;30B shows heater design in a single metal layer; 30C shows individualPCR channels overlaid with heater configurations; 30D shows individualPCR lanes.) In the embodiment shown, liquid sample is loaded without airbubbles as the lanes do not have any vents. Two or more Mux can beutilized for controlling all 96 PCR channels.

Such an arrangement lends itself to whole area imaging (e.g., by a CCD)for detection instead of optical based methods using diodes and lenses.

Example 9: Real-Time PCR

FIG. 31 shows a trace of real-time PCR carried out on multiple samplesin parallel with an apparatus and microfluidic network as describedherein. The PCR curves are standard plots that are representative offluorescence from 12 different PCR lanes as a function of cycle number.

The foregoing description is intended to illustrate various aspects ofthe present technology. It is not intended that the examples presentedherein limit the scope of the present technology. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A microfluidic substrate, comprising: a first PCR reaction chamber; asecond PCR reaction chamber; a first inlet, in fluid communication withthe first PCR reaction chamber; a second inlet, in fluid communicationwith the second PCR reaction chamber; a first set of microfluidic valvesconfigured to isolate the first reaction chamber from the first inlet;and a second set of microfluidic valves configured to isolate the secondPCR reaction chamber from the second inlet.
 2. A microfluidic substrate,comprising: a plurality of sample lanes, wherein each of the pluralityof sample lanes comprises a microfluidic network having, in fluidcommunication with one another: an inlet; a first valve and a secondvalve; a first channel leading from the inlet, via the first valve, to areaction chamber; and a second channel leading from the reactionchamber, via the second valve, to a vent.
 3. The microfluidic substrateof claim 2, additionally comprising: a third channel leading from theinlet to the reaction chamber, wherein a gate is positioned in the thirdchannel, and wherein the gate is configured to open the third channel topermit material from the reaction chamber to be removed from thecartridge via the inlet.
 4. The microfluidic substrate of claim 2,wherein each of the plurality of sample lanes is configured to amplifyone or more polynucleotides independently of the other lanes.
 5. Themicrofluidic substrate of claim 2, wherein each of the plurality ofsample lanes further comprises a bubble vent.
 6. The microfluidicsubstrate of claim 2, wherein the inlet is configured to accept samplefrom a pipette tip.
 7. The microfluidic substrate of claim 2, configuredto carry out real-time PCR in at least one of the reaction chambers. 8.The microfluidic substrate of claim 2, wherein the inlets of therespective plurality of sample lanes are spaced apart from one anotherto permit simultaneous loading from a multiple-pipette head dispenser.9. The microfluidic substrate of claim 2, wherein the first and secondvalves comprise a temperature responsive substance that melts uponheating and seals the reaction chamber.
 10. (canceled)
 11. (canceled)12. (canceled)
 13. (canceled)
 14. A method of carrying out PCRindependently on a plurality of polynucleotide-containing samples, themethod comprising: introducing the plurality of samples in to amicrofluidic cartridge, wherein the cartridge has a plurality of PCRreaction chambers configured to permit thermal cycling of the pluralityof samples independently of one another; moving the plurality of samplesinto the respective plurality of PCR reaction chambers; isolating theplurality of PCR reaction chambers; and amplifying polynucleotidescontained with the plurality of samples, by application of successiveheating and cooling cycles to the PCR reaction chambers.
 15. (canceled)16. (canceled)
 17. (canceled)